Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha

Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.     

Rapid kinetics of regulator of G-protein signaling (RGS)-mediated Galphai and Galphao deactivation. Galpha specificity of RGS4 AND RGS7

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits speeding deactivation. Galpha deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Galpha accompanying GTP hydrolysis and Galpha deactivation on the millisecond time scale. Basal k(cat) values for Galpha(o), Galpha(i1), and Galpha(i2) at 20 degrees C were similar (0.025-0.033 s(-1)). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of Galpha deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s(-1) at 3 microm, giving a catalytic efficiency of 1.7-2.8 x 10(6) m(-1) s(-1) for all three Galpha subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 x 10(6) m(-1) s(-1) toward Galpha(o), Galpha(i2), and Galpha(i1), respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Galpha subunits tested, but it is specific for Galpha(o) over Galpha(i1) or Galpha(i2). Furthermore, the specificity of RGS7 for Galpha(o) does not depend on N- or C-terminal extensions or a Gbeta(5) subunit but resides in the RGS domain itself.     

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3)

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.     

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.     

Engineering a V2 Vasopressin Receptor Agonist- and Regulator of G-Protein-Signaling-Sensitive G Protein

It is extremely difficult to detect guanine nucleotide exchange or hydrolysis stimulated by receptors which couple to G(s)alpha. Furthermore, G(s)alpha is largely resistant to the GTPase-activating properties of RGS proteins. Coexpression of the vasopressin V(2) receptor with a series of chimeric G protein alpha subunits in which the C-terminal 6-12 amino acids of G(i1)alpha were replaced with the equivalent sequence of G(s)alpha allowed robust vasopressin-stimulated [(35)S]GTPgammaS binding. Vasopressin did not stimulate the GTPase activity of fusion proteins between the V(2) receptor and either G(s)alpha or G(i1)alpha. However, it produced a concentration-dependent stimulation of V(max) for a V(2) receptor-G(i1)alpha/Gs6alpha fusion protein. This construct bound [(3)H]vasopressin with high affinity and this was competed by other ligands with rank order anticipated for the V(2) receptor. RGS1 enhanced vasopressin stimulation of V(2) receptor-G(i1)alpha/G(s)6alpha in a concentration-dependent manner. RGS-GAIP was substantially less potent. Enzyme kinetic analysis demonstrated that RGS1 increased both V(max) of the GTPase activity and the observed K(m) for GTP, consistent with RGS1 accelerating the rate of GTP hydrolysis of the chimeric G protein, whereas the agonist vasopressin accelerates guanine nucleotide exchange. This approach provides a sensitive assay for V(2) receptor agonist ligands and may be amenable to many other G(s)alpha-coupled receptors.     

The human delta opioid receptor activates G(i1)alpha more efficiently than G(o1)alpha

To assess the relative capacity of the human delta opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the alpha-subunits of either G(i1) or G(o1), containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in-frame with the C-terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [(3)H]naltrindole with high affinity. D-ala(2),D-leu(5) Enkephalin effectively inhibited forskolin-stimulated adenylyl cyclase activity in intact cells in a concentration-dependent, but pertussis toxin-insensitive, manner. The high-affinity GTPase activity of both constructs was also stimulated by D-ala(2),D-leu(5) enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to D-ala(2),D-leu(5) enkephalin was more than three times greater for G(i1)alpha than for G(o1)alpha. As the effect of agonist in both cases was to increase V:(max) without increasing the observed K:(m) for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of G(i1)alpha compared with G(o1)alpha. An equivalent fusion protein between the human mu opioid receptor-1 and G(i1)alpha produced a similar D-ala(2),D-leu(5) enkephalin-induced GTP turnover number as the delta opioid receptor-G(i1)alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G(i1)alpha.     

Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues

Hydrolysis of fluorescent GTP analogues BODIPY FL guanosine 5 '-O-(thiotriphosphate) (BGTPgammaS) and BODIPY FL GTP (BGTP) by Galpha(i1) and Galpha was characterized using on-line capillary electrophoresis (o) laser-induced fluorescence assays in order that changes in sub-strate, substrate-enzyme complex, and product could be monitored separately. Apparent k values (V /[E]) (max cat) steady-state and K(m) values were determined from assays for each substrate-protein pair. When BGTP was the substrate, maximum turnover numbers for Galpha and Galpha(i1) were 8.3 +/- 1 x 10(-3) and 3.0 +/- 0.2 x 10(-2) s(-1), respectively, and K(m) values were 120 +/- 60 and 940 +/- 160 nm. Assays with BGTPgammaS yielded maximum turnover numbers of 1.6 +/- 0.1 x 10(-4) and 5.5 +/- 0.3 x 10(-4) s(-1) for Galpha and Galpha(i1); K(m) values were 14 (o)(+/-)8 and 87 +/- 22 nm. Acceleration of Galpha GTPase activity by regulators of G protein signaling (RGS) was demonstrated in both steady-state and pseudo-single-turnover assay formats with BGTP. Nanomolar RGS increased the rate of enzyme product formation (BODIPY(R) FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199 -778% in pseudo-single-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38-81% by 40 mum YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that Galpha subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTPgammaS hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe.     

RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity

Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein alpha-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Ggamma-like (GGL) domain that mediates dimeric interaction with Gbeta(5). Gbeta(5)/R7 dimers stimulated steady state GTPase activity of Galpha-subunits of the G(i) family, but not of Galpha(q) or Galpha(11), when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gbeta(5)/R7 proteins revealed differences in potencies and efficacies toward Galpha-subunits of the G(i) family. Although all four Gbeta(5)/R7 proteins exhibited similar potencies toward Galpha(o), Gbeta(5)/RGS9 and Gbeta(5)/RGS11 were more potent GAPs of Galpha(i1), Galpha(i2), and Galpha(i3) than were Gbeta(5)/RGS6 and Gbeta(5)/RGS7. The maximal GAP activity exhibited by Gbeta(5)/RGS11 was 2- to 4-fold higher than that of Gbeta(5)/RGS7 and Gbeta(5)/RGS9, with Gbeta(5)/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gbeta(5)/RGS7 and Gbeta(5)/RGS9 inhibited Gbeta(5)/RGS11-stimulated GTPase activity of Galpha(o). Therefore, R7 family RGS proteins are G(i) family-selective GAPs with potentially important differences in activities.     

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

Polarity exchange at the interface of regulators of G protein signaling with G protein alpha-subunits

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and Galpha-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with Galpha(t) and Galpha(i1) was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of Galpha(i1) by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in Galpha(i1), this lysine was changed to glutamate for compensation. Galpha(i1)K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(i)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both Galpha(i1) and Galpha(i1)K210E mimicked the effect of alpha(2)-adrenoreceptor stimulation. Galpha(i1)K210E was sensitive to RGS16E89K and 10-fold more potent than Galpha(i1). Analogous mutants of Galpha(q) (Galpha(q)K215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type Galpha(q) was counteracted by wild type RGS4 but not by RGS4E87K. The activity of Galpha(q)K215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in Galpha(i) and Galpha(q) family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing Galpha-RGS specificities in living cells.     

Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4

Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.     

Isolation and characterization of a novel human RGS mutant displaying gain-of-function activity

Regulator of G protein signaling (RGS) proteins play a crucial role in the adaptation of cells to stimulation by G protein-coupled receptors via heterotrimeric G proteins. Alterations in RGS function have been implicated in a wide range of disease states, leading to many researchers focusing on controlling the action of these regulatory proteins. Previous studies have centered on reducing or inhibiting the action of RGS proteins, utilizing inactive mutants or small molecular RGS inhibitors. Here we describe the isolation and characterization of a novel human RGS4 mutant which displays enhanced or gain-of-function (GOF) activity. RGS4(S30C) demonstrates GOF activity both in an in vivo yeast-based signalling pathway and in vitro against the Galpha(o1) subunit contained in an alpha(2A)-adrenoreceptor-Galpha(o1)(C351I) fusion protein. Mutational analysis of serine 30 identified a number of alternative substitutions that result in GOF activity. GOF activity was retained upon transposition of the serine 30-cysteine mutation to the equivalent serine residue in human RGS16. As with previously identified GOF mutants, RGS4(S30C/S30F/S30K) demonstrate increased steady state protein levels, however these mutants also demonstrate enhanced GAP activity through an additional mechanism distinct from the increased protein content. The identification of human RGS mutants with GOF activity may provide novel therapeutic agents for the treatment of signaling-based diseases and the ability to transpose these mutations to other human RGS proteins extends their application to multiple pathways.     

RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity

The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.     

Mechanisms for reversible regulation between G13 and Rho exchange factors.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.     

Role of regulator of G protein signaling 2 (RGS2) in Ca(2+) oscillations and adaptation of Ca(2+) signaling to reduce excitability of RGS2-/- cells

Regulators of G protein signaling (RGS) proteins accelerate the GTPase activity of Galpha subunits to determine the duration of the stimulated state and control G protein-coupled receptor-mediated cell signaling. RGS2 is an RGS protein that shows preference toward Galpha(q).To better understand the role of RGS2 in Ca(2+) signaling and Ca(2+) oscillations, we characterized Ca(2+) signaling in cells derived from RGS2(-/-) mice. Deletion of RGS2 modified the kinetic of inositol 1,4,5-trisphosphate (IP(3)) production without affecting the peak level of IP(3), but rather increased the steady-state level of IP(3) at all agonist concentrations. The increased steady-state level of IP(3) led to an increased frequency of [Ca(2+)](i) oscillations. The cells were adapted to deletion of RGS2 by reducing Ca(2+) signaling excitability. Reduced excitability was achieved by adaptation of all transporters to reduce Ca(2+) influx into the cytosol. Thus, IP(3) receptor 1 was down-regulated and IP(3) receptor 3 was up-regulated in RGS2(-/-) cells to reduce the sensitivity for IP(3) to release Ca(2+) from the endoplasmic reticulum to the cytosol. Sarco/endoplasmic reticulum Ca(2+) ATPase 2b was up-regulated to more rapidly remove Ca(2+) from the cytosol of RGS2(-/-) cells. Agonist-stimulated Ca(2+) influx was reduced, and Ca(2+) efflux by plasma membrane Ca(2+) was up-regulated in RGS2(-/-) cells. The result of these adaptive mechanisms was the reduced excitability of Ca(2+) signaling, as reflected by the markedly reduced response of RGS2(-/-) cells to changes in the endoplasmic reticulum Ca(2+) load and to an increase in extracellular Ca(2+). These findings highlight the central role of RGS proteins in [Ca(2+)](i) oscillations and reveal a prominent plasticity and adaptability of the Ca(2+) signaling apparatus.     

Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTPgammaS-stimulated Galpha(11) signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10-20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-betat activity than of GTPgammaS-promoted PLC-betat activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4beta-phorbol 12beta-myristate 13alpha-acetate and, to a lesser extent, by the P2Y(2) receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTPgammaS-stimulated PLC-betat activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y(1) receptor and Galpha(q)betagamma. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.     

Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10).

Heterotrimeric G proteins are involved in the transduction of hormonal and sensory signals across plasma membranes of eukaryotic cells. Hence, they are a critical point of control for a variety of agents that modulate cellular function. Activation of these proteins is dependent on GTP binding to their alpha (Galpha) subunits. Regulators of G protein signaling (RGS) bind specifically to activated Galpha proteins, potentiating the intrinsic GTPase activity of the Galpha proteins and thus expediting the termination of Galpha signaling. Although there are several points in most G protein controlled signaling pathways that are affected by reversible covalent modification, little evidence has been shown addressing whether or not the functions of RGS proteins are themselves regulated by such modifications. We report in this study the acute functional regulation of RGS10 thru the specific and inducible phosphorylation of RGS10 protein at serine 168 by cAMP-dependent kinase A. This phosphorylation nullifies the RGS10 activity at the plasma membrane, which controls the G protein-dependent activation of the inwardly rectifying potassium channel. Surprisingly, the phosphorylation-mediated attenuation of RGS10 activity was not manifested in an alteration of its ability to accelerate GTPase activity of Galpha. Rather, the phosphorylation event correlates with translocation of RGS10 from the plasma membrane and cytosol into the nucleus.     

Dominant-negative inhibition of pheromone receptor signaling by a single point mutation in the G protein alpha subunit

In yeast, two different constitutive mutants of the G protein alpha subunit have been reported. Gpa1(Q323L) cannot hydrolyze GTP and permanently activates the pheromone response pathway. Gpa1(N388D) was also proposed to lack GTPase activity, yet it has an inhibitory effect on pheromone responsiveness. We have characterized this inhibitory mutant (designated Galpha(ND)) and found that it binds GTP, interacts with G protein betagamma subunits, and exhibits full GTPase activity in vitro. Although pheromone leads to dissociation of the receptor from wild-type G protein, the same treatment promotes stable association of the receptor with Galpha(ND). We conclude that agonist binding to the receptor promotes the formation of a nondissociable complex with Galpha(ND), and in this manner prevents activation of the endogenous wild-type G protein. Dominant-negative mutants may be useful in matching specific receptors and their cognate G proteins and in determining mechanisms of G protein signaling specificity.     

RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.     

Regulators of G protein signaling 6 and 7. Purification of complexes with gbeta5 and assessment of their effects on g protein-mediated signaling pathways.

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein beta subunits, and certain biochemical properties of RGS6/beta5 and RGS7/beta5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gbeta5 subunit (but not Gbetas 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/beta5 in that they act as GTPase-activating proteins specifically toward Galpha(o). Unlike conventional G(betagamma) complexes, RGS6/beta5 and RGS7/beta5 do not form heterotrimeric complexes with either Galpha(o)-GDP or Galpha(q)-GDP. Neither RGS6/beta5 nor RGS7/beta5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-beta1 or -beta2. However, the RGS/beta5 complexes inhibited beta(1)gamma(2)-mediated activation of phospholipase C-beta2. RGS/beta5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G(i) and G(o) by binding to phospholipase C and stimulating the GTPase activity of Galpha(o).